At least 55,000 chemicals are presently produced in the United States. Over 2,000 new chemicals are introduced into the market each year. Very few of these chemicals have been comprehensively tested for acute or chronic toxicity. For example, less than 1 percent of commercial chemicals have undergone complete health hazard assessment.
The Environmental Protection Agency ("EPA") has the authority to require toxicological testing of a chemical prior to commercial production, but that authority is rarely invoked. Less than 10 percent of new chemicals are subjected to detailed review by the EPA. In the interest of cost and speedy access to the market, the EPA often uses the toxicity of previously tested homologous compounds to gauge the toxicity of a new chemical.
The potential toxicity of new drugs is monitored by the Food and Drug Administration ("FDA"). For a New Drug Application (NDA), the FDA typically requires a large battery of toxicity, carcinogenicity, mutagenicity and reproduction/fertility tests in at least two species of live animals. These tests are required to last up to one year. The costs involved in completing these tests is enormous. For example, a typical 90-day exposure toxicity test in rats costs approximately $100,000. A two year toxicity test in rats costs approximately $800,000 [Casarett and Doull's Toxicology, 4th Edition, M. O. Amdur et al., eds. Pergamon Press, New York, N.Y., p. 37 (1991)].
Besides cost, animal testing also presents disadvantages in terms of time, animal suffering and accuracy. Typical toxicity tests are divided into three stages: acute, short term and long term. Acute tests, which determine the LD.sub.50 of a compound (the dose at which 50% of test animals are killed), require some 60-100 animals and a battery of tests for determining LD.sub.50, dose-response curves and for monitoring clinical end points, other than death. Short term tests usually involve at least 24 dogs and 90 rats and last from 90 days in rats to 6-24 months in dogs. Body weight, food consumption, blood, urine and tissue samples are frequently measured in the short-term tests. In addition, dead animals are subjected to post-mortem examinations. Long term tests are similar to short term tests, but last 2 years in rats and up to 7 years in dogs or monkeys.
Animal testing has come under criticism by animal rights activists and the general public because of the severe suffering inflicted on the animals. Moreover, recent evidence calls into question the accuracy of animal testing. For example, variables, such as animal diet, may impair the predictability of animal tests in determining carcinogenic properties [P. H. Abelson, "Diet and Cancer in Humans and Rodents", Science, 255, p. 141 (1992)]. And prior determinations on dioxin toxicity, based on guinea pig testing, are now being reevaluated [B. J. Culliton, "U.S. Government Orders New Look At Dioxin", Nature 352 p. 753 (1991); L. Roberts, "More Pieces in the Dioxin Puzzle", Research News, October, 1991, p 377]. It is therefore apparent that there is an urgent need for a quick, inexpensive and reliable alternative to toxicity testing in animals.
Several short-term alternative tests are available. For example, the Ames Assay detects carcinogens which cause genetic reversion of mutant strains of Salmonella typhimurium. However, the Ames Assay cannot detect either non-mutagenic carcinogens or non-carcinogenic toxins. The yeast carcinogen assay system described in U.S. Pat. No. 4,997,757 overcomes some of the drawbacks of the Ames Assay, but is still not able to detect non-carcinogenic toxins. Both of these assays are designed to detect alterations and mutations at the DNA level only. Therefore, those prior art tests cannot detect direct damage to proteins or lipid membranes, nor inhibitors of DNA synthesis. Moreover, none of the short-term tests presently employed yields any information about the cellular mechanism by which a carcinogen, mutagen or toxin exerts its effect. Therefore, these prior art assays also do not reveal any information that would be helpful in selecting a counteracting agent or antidote to a compound found to be toxic.